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WO2015052046A1 - Procédé in vitro pour déterminer sans marquage un type de cellules - Google Patents

Procédé in vitro pour déterminer sans marquage un type de cellules Download PDF

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Publication number
WO2015052046A1
WO2015052046A1 PCT/EP2014/070951 EP2014070951W WO2015052046A1 WO 2015052046 A1 WO2015052046 A1 WO 2015052046A1 EP 2014070951 W EP2014070951 W EP 2014070951W WO 2015052046 A1 WO2015052046 A1 WO 2015052046A1
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WO
WIPO (PCT)
Prior art keywords
cell
determining
compartment
cells
height profile
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/EP2014/070951
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German (de)
English (en)
Inventor
Oliver Hayden
Oliver Schmidt
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Siemens AG
Siemens Corp
Original Assignee
Siemens AG
Siemens Corp
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Siemens AG, Siemens Corp filed Critical Siemens AG
Priority to CN201480055813.9A priority Critical patent/CN105659068B/zh
Priority to EP14781497.4A priority patent/EP3042177B1/fr
Priority to US15/024,413 priority patent/US10408735B2/en
Publication of WO2015052046A1 publication Critical patent/WO2015052046A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N15/1429—Signal processing
    • G01N15/1433—Signal processing using image recognition
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01B—MEASURING LENGTH, THICKNESS OR SIMILAR LINEAR DIMENSIONS; MEASURING ANGLES; MEASURING AREAS; MEASURING IRREGULARITIES OF SURFACES OR CONTOURS
    • G01B11/00—Measuring arrangements characterised by the use of optical techniques
    • G01B11/02—Measuring arrangements characterised by the use of optical techniques for measuring length, width or thickness
    • G01B11/06—Measuring arrangements characterised by the use of optical techniques for measuring length, width or thickness for measuring thickness ; e.g. of sheet material
    • G01B11/0608—Height gauges
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/01—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials specially adapted for biological cells, e.g. blood cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/01—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials specially adapted for biological cells, e.g. blood cells
    • G01N2015/012—Red blood cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/01—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials specially adapted for biological cells, e.g. blood cells
    • G01N2015/016—White blood cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N15/1434—Optical arrangements
    • G01N2015/1454—Optical arrangements using phase shift or interference, e.g. for improving contrast
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N2015/1493—Particle size

Definitions

  • the invention relates to a method for the label-free determination of a cell type of a cell using a microscopy device.
  • the determination of cells and their assignment to a cell type is of great importance in cytology.
  • cytology In, for example, a hematological examination, cellular blood components, ie erythrocytes, platelets and white blood cells, are determined and quantified.
  • WBC White blood count
  • the white cells are differentiated into granular cells (neutrophils, eosinophils, basophils) and non-granular cells (lymphocytes, monocytes In addition to the granularity, the cells also differ in terms of fragmentation of the nucleus (without fragmentation: mononuclear cells, ie lymphocytes and monocytes, polymorphonuclear cells:
  • Eosinophils, basophils and neutrophils Eosinophils, basophils and neutrophils
  • cell size For the differentiation of granular cells, in particular eosinophilic and basophilic granulocytes, a staining is used.
  • the evaluation of the cell populations usually takes place by fully automated hematology analyzers or by microscopy.
  • Fully automated analyzers müs- sen the populations of fixed algorithms analyze (by means of, for example impedance, scattered light and Absorpti ⁇ ons horren). However, this often leads to error messages being displayed, for example, in the case of pathological samples.
  • microscopy is usually used as a validation method for cells incorrectly determined by the hematology analyzer. This step is costly and time-consuming, since, in addition to sample preparation, microscopy and pie and other manual work also requires manual assessment.
  • cell volume In addition to the morphology of the cell, it may also be important to determine cell volume. This is usually done only in non-granular red blood cells and platelets due to the scattering by granules and polymorphic nuclei. Therefore, this is not usually done for white blood cells.
  • a hematology analyzer can be dispensed with and instead each individual cell can be microscoped. This he ⁇ laubt it, algorithms blood count regardless of set evaluation and to determine quantitatively flags. Disadvantage of this approach, however, is the lower sample throughput than in a hematology analyzer and the ongoing effort to immobilize the cells on slides and to dye. Moreover, these dyeings have only a limited reproducibility and show a high dependence on
  • An object of the invention is therefore to provide a more efficient and robust method for differentiating cells.
  • the object is achieved by the method according to the invention, the cell analysis device according to the invention and the Microscoping solved according to the independent claims.
  • Advantageous developments of the invention are given by the dependent claims.
  • the in-vitro method according to the invention for the label-free determination of a cell type of a cell in a biological sample is carried out with the aid of a microscope device and a cell analysis device.
  • Microscopy device is a device that can be considered greatly enlarged with the very small objects, and for example, an optical microscope, a scanning electron ⁇ electron microscope, a phase contrast microscope, a digital ho ⁇ lographisches microscope or a microscope with an ultra ⁇ sound sensor (eg, includes an acoustic microscope ).
  • an optical microscope e.g., an optical microscope, a scanning electron ⁇ electron microscope, a phase contrast microscope, a digital ho ⁇ lographisches microscope or a microscope with an ultra ⁇ sound sensor (eg, includes an acoustic microscope ).
  • Microscopy detects a height profile of the
  • a height profile is a profile that describes the position and height of the cell, thus providing topographic information.
  • the cell analysis device ie a device or a device component which is suitable for electronic data processing and is set up to process the data of a height profile, carries out the following steps in the method according to the invention:
  • Determining the cell type of the cell on the basis of the determined quantitative cell characteristic.
  • a cell compartment is understood as meaning the cell area which is surrounded by a biomembrane.
  • a mitochondrion, a nucleus, a vacuole, cytoplasm or granules is therefore understood hereinafter, wherein a
  • Granules so a visible, granular storage of the Cell, as a cell compartment and several granules are understood as meh ⁇ rere cell compartments.
  • the invention shown SSE process provides a robust and label-free method for determining a cell type.
  • the method of the invention is equally suitable for determining a cell type of a cell as well as for differentiating cells of a heterogeneous cell suspension.
  • the determination of the cells is not impaired by the physical properties of the staining material.
  • rounding buffers for example a diluted SDS buffer solution
  • the method allows following the withdrawal of a dyeing process ⁇ a high sample throughput and allows for a qualitative and / or quantitative determination of cells independent of set evaluation algorithms or binary status indicators. This allows microscopy with a high sample throughput and can be dispensed with conventional hematology analyzers. Is carried out according to a further embodiment of the erfindungsge ⁇ MAESSEN method, determining the predetermined quantitati ⁇ ven cell feature based of the height profile, then this leads to a higher resolution and thereby a more precise determination of the cell type. Alternatively, an additional quantitative cell feature can be used, which is not determined based on the height profile but, for example, based on intensity, polarization or fluorescence to determine the cell type.
  • a further, particularly preferred improvement of Bestim ⁇ mung the cell characteristics can be achieved in that the determination of the cell compartment by determining egg Cell volume, determining a first Zellkompartiments, for example, the nucleus, and determining a further Zellkompartiments done. Determining a Zellkomparti ⁇ ments may include, for example, a segment the cell compartments management. This represents a particularly preferred exporting ⁇ approximate shape of the inventive process. The region of the first compartment can be neglected, for example, during the determination of the second compartment.
  • the quantitative cell feature in a further exporting ⁇ approximate shape of the inventive method for example, a cell volume, a volume of the cell compartment, a Zellflä ⁇ che, a surface of the cell compartment, a ratio of cell volume to a cell nucleus volume, a ratio of a cell area to a cell nucleus area has a diameter of
  • Zellkompartiments a roundness of the cell compartment, a circumference of the cell compartment, a number of Zellkomparti ⁇ ment and / or a statistical feature of a cell characteristic, based on the respective height profiles of several cells
  • the quantitative cell shopping times ⁇ describes a statistical feature of a cellular characteristic which is determined by the respective height profiles a plurality of cells and / or from the height profiles of several cell compartments. This allows a higher resolution in the classification of the cells and a better distinguishability, especially between different cells, which are difficult to differentiate due to conventional methods.
  • the statistical feature is preferably a variance of the diameter of the same cell compartments, for example a variance of the
  • a statistical characteristic on the basis of exemplary meh ⁇ cells can be used.
  • the mean diameter of the nucleus of a plurality of, for example, white blood cells may be set as cells in relation to the diameter of the nucleus of the cell under study.
  • the considered the maynpro ⁇ fil comes to the detection of the cell characteristics are used, however, additional information can be acquired in other embodiments, which additionally available ste ⁇ hen in response to the Mirkroskopievons.
  • additional information can be acquired in other embodiments, which additionally available ste ⁇ hen in response to the Mirkroskopievons.
  • the intensity distribution, polarization or fluorescence contrast can additionally be used to enable a better differentiation.
  • Cell suspension analyzes for example in a differential blood picture in which, for example, white blood cells are distinguished under ⁇ .
  • the acquisition of the height profile is preferably carried out by superimposing an object wave with a reference wave, recording a resulting interferogram and / or a mathematical reconstruction, for example by means of phase contrast microscopy.
  • This particularly advantageous execution ⁇ of the method according to the invention is particularly suitable for determining quantitative characteristics, above all, it favors a label-free differential blood analysis.
  • the images obtained by the phase contrast microscopy are particularly well reproducible and have a high resolution ⁇ on solution.
  • the height profile is thus determined by means of digital holographic microscopy, Interpherenzphasenmikroskopie and / or quantitati ⁇ ver phase microscopy.
  • digital holographic microscopy allows a particularly high axial resolution, ie in the direction of the optical axis of the microscope.
  • a digital holographic microscopy allows a resolution of up to ln in the z-direction. This corresponds to a precision which is higher by a factor of 100-1000 than with other known light microscopic methods (eg confocal microscope). Due to the precise determination of the height profile thus a more accurate determination of the cell type is possible.
  • Another advantage is achieved when the process is carried out with one or more unstained and / or undried cells. Since the vitality of the cells through which it ⁇ -making proper procedures not compromised, determined by the method cells can advertising continue to be used after completion of the assay for the analysis of consequences.
  • the method according to the invention can be used to phenotype the cell with a marker. ker and / or expressing a predetermined receptor for further assigning the cell. This allows for a more detailed molecular investigation of ⁇ be voted cell.
  • a particular relevance invention receives procedural ⁇ ren which, when it is performed according to another embodiment with reference to a whole blood sample and / or the biological sample comprises blood cells, leukocytes, eosinophils and / or basophils. This is mainly used in the determination of a blood count in hematology.
  • determining the cell type may be for differentiating neutrophils, eosinophils, basophil lymphocytes, and / or monocytes, and / or determining cell functionality, in particular determining an activation state of a cell, e.g. of a platelet, and / or detection of a cell, e.g. a deformed erythrocyte and / or a pathogen-affected cell.
  • the determining of the cell type can include determining ei ⁇ nes cell stage of the cell, in particular for differential renzieren a cell age, physiological or morphological state of the cell or an activity state of the cell. This makes it possible to record a picture sequence of, for example, activation processes of a cell.
  • the above object is also achieved by a
  • Cell analysis device for example a microchip or a microcontroller, which is adapted to perform one or more of the above-described embodiments of the method.
  • the above object is also achieved by a microscope device for mark-free determination of a cell type of a cell of a biological sample, comprising Embodiment of the above cell analysis device.
  • the microscope device comprises a digita ⁇ les holographic microscope.
  • FIG. 3 shows a schematic representation of determining a cell compartment and of determining ei ⁇ nes predetermined quantitative cell feature according to one embodiment of the erfindungsge ⁇ MAESSEN method
  • 4a, 4b is a schematic sketch for determining the
  • Cell type of cell based on determined quantitative cell characteristics.
  • a biological sample may comprise, for example, a sample of animal or plant cells, bacterial cells and / or unicellular organisms.
  • this is a whole blood sample comprising, for example, blood cells 10, for example leucocytes, eosinophilic granulocytes or basophilic granulocytes.
  • blood cells 10 for example leucocytes, eosinophilic granulocytes or basophilic granulocytes.
  • the different exemplary cell types are both in the 1, as well as in FIGS.
  • the white blood cells include
  • granular cells 10 for example eosinophilic granulocytes 10, basophilic granulocytes 10 and neutrophilic granulocytes 10.
  • the non-granular cells 10 include lymphocytes 10 and monocytes 10.
  • cell 10 is exemplified by each cell type.
  • the exemplary non ⁇ granular cells 10 each have a cell nucleus 12th
  • the remaining exemplary granulocytes 10 have irregularly lobed cell nuclei 12, ie they are
  • the granular cells 10 have in common that they have, for example, several granules 12 "in their interior. For clarity, in FIG 1 in these cell h ⁇ len only some of the granules 12 '' marked with reference numerals.
  • the white blood cells 10 also differ in cell size.
  • Alternative cell compartments 12 are, for example, a mitochondrium, a vacuole, a cytoplasm or an endoplasmic reticulum.
  • the 2 shows schematically the principle of an embodiment ⁇ example of the method according to the invention is shown.
  • the 2 shows a microscope device 14, which is to be ⁇ sets, a topography, that is, to determine a height information of a cell precisely.
  • the resolution of the considered formati ⁇ on is at least 1 micron, preferably 100 nanometers ter or less than 100 nanometers, more preferably 10 nanometers or less than 10 nanometers.
  • the microscope device 14 comprises, as in the example of FIG. 2, a digital holographic microscope.
  • the digital holographic microscopy also Interfe ⁇ ence phase microscopy called, is characterized in that it can detect both the intensity and the phase of a Whether ⁇ jekts quantitatively in a receptacle. For this will not, as is customary in the microscopy, was added a Intensticiansvertei ⁇ lung, which results from the absorption and scattering of light on the object, but a wave front, which results from the superposition of an object and a reference wave. From this, the intensity and phase information of the object can be reconstructed with the aid of a computer.
  • This microscopy method is suitable for the examination of biological samples, since they are substantially transparent to visible light without further sample preparation and therefore have a low contrast in a conventional microscope.
  • phase information it is possible to take very precise about it ⁇ the morphology of the cell and make a differentiation of the cell using this information.
  • the DHM offers the advantage that the phase can be determined quantitatively. Accordingly, one also speaks of quantitative phase contrast microscopy. Only by quantifying the phase is a reproducible reconstruction of the height profile of the object possible and only then can an automated determination of the cell type take place.
  • phase shift of the light determined by the optical path length through the object is, ie, both the geometric thickness of the object and the refractive index plays a role.
  • phase information of the refractive index of the sample must be known.
  • the Pha ⁇ seninformation be recorded at different wavelengths and thus, the influence of the refractive index
  • phase information is uniquely determined only to a phase angle of 360 °.
  • a measured phase shift of, for example, 10 ° may occur in
  • a height profile 18 of a respective cell 10 is detected (method step S1), which is e.g. is determined from a digital hologram height profile or an interferogram.
  • Microscope device 14 e.g. an acoustic microscope, the height profile can be determined from a sonogram.
  • method step S1 comprises a recording of a hologram, an iterative reconstruction of the phase and / or intensity information of the object and / or a conversion of the phase information into a height profile.
  • Detecting the height profile 18 comprises He ⁇ hold a cell contour, so a height profile of the cell Surface in relation to the support plate 16.
  • the support plate 16 may include, for example, a slide or another, common to the expert substrate for microscopic Un ⁇ tersuchungen.
  • a continuous strip for example consisting of a polymer, which is pulled through continuously under the objective.
  • FIG. 2 additionally shows a cell analysis device 13, which performs the method steps S2 and S3.
  • the exemplary cells 10 are determined, for example a differentiation of white blood cells, which is based, for example, on a reconstructed phase information of the cells 10.
  • first one or more cell compartments 12 of the cell 10 are determined on the basis of the detected height profile 18 (S2).
  • the left-hand height profile 18 of the first exemplary cell 10 exhibits two pronounced elevations, which, for example, indicate the presence of a polymorphic nucleus 12 '.
  • three smaller elevations of the height profile 18 can be seen, pointing to three granules 12 ''.
  • the vertical profile 18 of the second cell 10 to a mononuclear ⁇ arene nucleus 12 'and four granules 12''out.
  • the determining a Budapest ⁇ voted quantitative cell trait is carried out a number of one of the cell compartments 12 or a diameter of one of the cell compartments 12, based on the determined cell compartments ments 12. It follows that determining the cell type of the cell 10 using of the qualitative cell characteristic (S3). In the example of FIG. 2, for example, an eosinophilic granulocyte 10 (left in the picture) and a basophilic granulocyte 10 (right in the picture) are determined on the basis of the height profile 18.
  • the 3 shows a schematic representation of a imple ⁇ proceedings of determining the cell compartment 12 (S2).
  • the top of the image, an exemplary height profile 18 is to a cell h ⁇ le 10 shown on a support plate sixteenth In a first Step, for example, a cell volume of the cell 10 is determined for this purpose.
  • Methods for determining the cell volume based ei ⁇ nes height profile are known to those skilled in the art (for example, integration or summation of the height over the entire area of the cell).
  • the surface of the cell can be done for example on the basis of a threshold value in the absorption ⁇ contrast or height profile.
  • step S2a subsequent segmenting one of the cell compartments 12, shown in the present example the cell ⁇ core 12 'as the first cell compartment 12.
  • the Seg ⁇ menting is carried out based on the height information for this cell compartment 12.
  • the segmenting can beispielswei- se by providing an interferogram by a phase-contrast method successes.
  • providing the height profile may include, for example, lasing, screening, or ultrasound-bound scanning.
  • the middle illustration of Figure 3 shows two auxiliary lines 20, with 20 narrow the two vertical guides the exemplary first cell ⁇ compartment 12 'descriptive segment of the height profile 18 and the horizontal auxiliary line 20 a marked before ⁇ certain threshold, for example, a value of 50 percent of the height of the first cell compartment 12 'describes.
  • ⁇ certain threshold for example, a value of 50 percent of the height of the first cell compartment 12 'describes.
  • For the remaining part of the cell 10 is calculated at ⁇ play, of the mid-height and, for example, the ma ⁇ imum amount of the segmented region that threshold in order to determine, for example, a diameter of the cell compartment 12 or the other cell compartments 12 and the number thereof.
  • ⁇ play of the mid-height
  • the ma ⁇ imum amount of the segmented region that threshold in order to determine, for example, a diameter of the cell compartment 12 or the other cell compartments 12 and the number thereof.
  • segmenting the first cell compartment 12 one of ordinary skill in the art may use
  • an angle describing a tilted plane of the carrier plate 16 can be taken into account in the calculation.
  • the area of the first compartment is no longer considered. If, for example, the first cell association 12 is the nucleus 12 'and the second cell association is a granule 12 "or more granules 12", it can be assumed, for example in a second segmentation, that no granules exist at the nucleus 12' 12 '' are.
  • a further contrast mechanism can be drawn in by determining a further baseline 22.
  • This further base line 22 may, for example, represent a further threshold value for a further cell compartment 12.
  • a threshold value is, for example, ge ⁇ chooses to one or more granules parti elements 12 as Zellkom- 'to determine, for example,'.
  • a number, a diameter and / or an average size of granules can be determined.
  • a number of cell compartments 12 "per cell area can be determined.
  • a ratio of a cell area to an area of the cell nucleus by way of example determined by a number of a cell nucleus, a ratio of a cell area to an area of the cell nucleus, an average diameter of granule and a variance in diameter of the granules quan ⁇ tative cell characteristics.
  • a cell volume, a volume of the cell compartment, a cell surface, a surface of the Zellkomparti ⁇ ments a ratio of a cell volume can be detected at a Zellkernvo ⁇ lumen by quantitative cell characteristics.
  • FIGS. 4a and 4b show the determination of the cell type of the cell 10 on the basis of the determined quantitative cell characteristic.
  • FIG. 4A shows a matrix for classifying a cell 10 or more cells 10 whose ordinate Y ⁇ example, a number of Referenzkompartiments 12, for example, a laxity of a nucleus 12 ', and the abscissa X denotes, for example, a ratio of a cell surface to a surface of the cell nucleus.
  • additional dashed lines 20 are shown. If the cells 10 of the exemplary cell mixture according to the above exemplary quantitative cell characteristics belongs to the matrix, it can be the loading of existing lymphocytes ⁇ agree 10, 10 and monocytes
  • a further cluster is formed, for example, by basophilic granulocytes 10 and eosinophilic granulocytes 10.
  • step S3a can be distinguished clearly at play ⁇ liable granulocytes from one another.
  • An improvement in the distinction between the exemplary basophilic granulocytes 10 and eosinophilic granulocytes 10 can be achieved by further classification according to further quantitative cell characteristics.
  • FIG. 4b Particularly advantageous has proven to when the exemplary two other quantitative cell characteristics describes a statistical characteristic of a cell watch ⁇ times, which is determined by the respective height profiles of multiple cells 10.
  • a variance of a diameter of the granules is plotted on the ordinate Y, while a mean diameter of the granules is plotted on the abscissa X.
  • the matrix which is also intended here to facilitate classification by dashed lines, clearly shows a separation of the cells 10.
  • the exemplary embodiment described above can be carried out, for example, to determine the cell functionality of the cell 10, in particular as determining the functionality of, for example, a white blood cell and / or a non-peripheral blood cell and / or a pathogenously affected cell 10.
  • the functionality of a white blood cell can determined, for example, by the change in volume, the change in the number of granules and / or the change in the cell nucleus the.
  • non-peripheral blood cells 10 are, for example, bone marrow cells and immature cells.
  • the method described may, for example, in the context ei ⁇ ner lavage in, for example, an infection or a lower investigation of cerebrospinal fluid in an infection done. This also allows label-free detection of a Parasitic ⁇ ren infection, such as the detection of malaria parasites in red blood cells in defined cell cycle stages, with early diagnosis is desired.
  • determining the cell type may be a determination of cell stage of the cell 10 include, in particular for differentiating a cell age example ⁇ example the age of granular neutrophils, wherein, for example, a shift in the classification ⁇ matrix at, for example young rod-shaped neutrophils compared to mature round neutrophils occurs.
  • a physiological or a morphological state of a cell 10 can be determined, for example in the context of a detection of a pathological blood finding by over-segmented
  • Nuclei of neutrophilic granulocytes due to, for example, an iron deficiency Another example is the detection of, for example, deformed erythrocytes, for example echinocytes in, for example, the presence of sickle cell anemia. Another example is determining the cell type within a size distribution of erythrocytes to distinguish
  • Reticulocytes so young erythrocytes.
  • Differentiation of, for example, quiescent and / or activated platelets by a change in morphology can also be carried out in the context of the described method according to the invention.
  • An example ⁇ -like approach to create a Differenzialblut- image includes, for example, determining a cell size, egg ⁇ nes maturation state of the cell, so an age of the cell, a core plasma ratio, the detection of
  • Microscopy detected height profile 18 at least one cell compartment 12 of the cell 10 to detect and using the determined cell compartment 12 and / or the height profile 18 to determine a quantitative cell feature.
  • an automatable and Markie ⁇ approximately free microscopy technique is used to Probenvorberei ⁇ processing steps, such as a staining cells 10 to minimize, and also a label-free determination of a quantitative cell feature to enable, for example, a volume determination of the cell 10.
  • an interference phase microscopy and digital holographi ⁇ specific microscopy is preferably used, which enables, for example, a label-free differential blood analysis, for example, white blood cells 10, as it used to differentiate between, for example, eosinophil and basophilic granulocytes 10, for example, the granularity, nuclear morphology, and the cell ⁇ volumes.
  • this represents the greatest challenge in the differentiation of, for example, white blood cell populations.
  • a cell compartment 12 for example a cell nucleus 12 ', is segmented.
  • a threshold value is calculated (for example, 50 percent) in order, for example, a diameter for the case ⁇ play of the granules and their Number to determine (FIG 3).
  • the differentiation can then be derived from this parameter (FIG. 4a, FIG. 4b).
  • a size and a shape of a cell nucleus 12 ' can be used to distinguish, for example, lymphocytes, monocytes, neutrophils, eosinophils and basophils (FIG. 4a).
  • the following quantitative cell characteristics can be used for this purpose: a ratio of cell 10 volume to cell nucleus 12 ', a number and / or volume of, for example, polymorphic cell nuclei 12', information about number, diameter and size distribution, for example Granules, which are then used to distinguish, for example, eosinophilic and basophilic granulocytes (see FIG 4b).
  • Interference phase microscopy also known as digital holographic microscopy (DHM)
  • DHM digital holographic microscopy
  • Interference phase microscopy is characterized in that it can detect both an intensity and a phase of an object quantitatively in a photograph.
  • an image of the object is not, as is customary in microscopy detected, but a wavefront which results from the over ⁇ storage of an object and a reference wave. This can be reconstructed with the aid of a cell analysis device, for example a computer, an intensity and phase information of the object.
  • This microscopy method is suitable for examination of biological samples, as these without further Probenvorbe ⁇ reitung are substantially transparent to visible light and therefore have a lower contrast in a conventional microscope.
  • NEN phase information
  • Differentiation of, for example, white blood cells based on, for example, reconstructed phase information from multiple cells 10 may be carried out without requiring staining of the cells 10 and thereby working with fresh, non-dried cells. This avoids unnecessary artifacts caused by differential handling of the samples during drying and dyeing.
  • the images are distinguishable reproduction ⁇ zierbarer and lighter rechnerge ⁇ supported by an automatic image processing. The number of process steps is minimized. This saves costs and time and enables reliable and faster diagnostics.
  • pre analytics can be so opposite to the hematology analyzers on the employment of for example Rounding
  • Buffers are omitted (ie highly diluted SDS buffer solution). Such would lead to the rounding of cells, although for the scattered light measurement
  • Flow cytometer is suitable, but at the same time leads to artefacts.
  • erythrocytes are very sensitive and form echinocytes.
  • the invention comprises the following aspects: 1. A method for differentiation of blood cells, based on
  • Apparatus for differentiation of blood cells consisting of a) a digital holographic microscope,
  • phase information for example to determine the volume of an entire cell 10 or individual segments of the cell 10,
  • the exemplary blood corpuscles to be distinguished are, for example, leucocytes,
  • the exemplary leucocytes to be differentiated are, for example, eosinophils and basophils, for example the difference between cell volume and cell volume is used to differentiate,
  • a discrimination is achieved, for example in a multi-stage process, for example, first on the basis of the cell nucleus ⁇ volume and then, for example, based on a
  • the invention includes the following aspects: microscopy of, for example, multiple cells 10, for example, directly in a blood plasma, preferably near in vivo conditions; for example, citrated blood, heparinized blood, EDTA blood for the prevention of blood clotting,
  • activation of, for example, granular cells 10 without marking can be microscoped (for example the modification of, for example, granules 12 '', which are released, for example, by granulocytes 10),
  • erythrocytes for example echinocytes, sickle cell anemia
  • erythrocytes to distinguish reticulocytes (young erythrocytes)
  • non-peripheral blood cells e.g., bone marrow; immature cells), lavage (In Stammio ⁇ nen), cerebrospinal fluid (infections),
  • a marker-free determination of a parasitic infection for example malaria, ie parasites in erythrocytes in defined cell cycle stages, early diagnosis desired.
  • a current approach in the differential blood picture can thus be performed label-free with, for example, an interference microscope 14: for example, cell size, maturation, nuclear-plasma ratio, cytoplasmic inclusions, chromatin structure.
  • an interference microscope 14 for example, cell size, maturation, nuclear-plasma ratio, cytoplasmic inclusions, chromatin structure.

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Abstract

L'invention concerne un procédé in vitro pour déterminer sans marquage un type de cellules (10) dans un échantillon biologique, un dispositif microscopique (14) détectant (S1) un profil en hauteur (18) de la cellule (10) par rapport à une plaque de support (16). Le procédé est caractérisé par les étapes réalisées au moyen d'un dispositif d'analyse cellulaire (13) consistant à : déterminer un compartiment cellulaire (12, 12', 12'') de la cellule (10) en fonction du profil en hauteur (18, S2) détecté, à déterminer une caractéristique cellulaire quantitative prédéterminée en fonction du compartiment cellulaire (12, 12', 12'', S3) déterminé ; et déterminer le type de la cellule (10) en fonction de la caractéristique cellulaire (S3) quantitative déterminée. L'invention concerne également un dispositif d'analyse cellulaire (13) correctement configuré et un dispositif microscopique (14) correspondant.
PCT/EP2014/070951 2013-10-09 2014-09-30 Procédé in vitro pour déterminer sans marquage un type de cellules Ceased WO2015052046A1 (fr)

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CN201480055813.9A CN105659068B (zh) 2013-10-09 2014-09-30 用于细胞的细胞类型的无标识确定的体外方法
EP14781497.4A EP3042177B1 (fr) 2013-10-09 2014-09-30 Procédé in vitro pour déterminer sans marquage un type de cellules
US15/024,413 US10408735B2 (en) 2013-10-09 2014-09-30 In vitro method for the label-free determination of a cell type of a cell

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DE102014200911.1A DE102014200911A1 (de) 2013-10-09 2014-01-20 In-Vitro-Verfahren zum markierungsfreien Bestimmen eines Zelltyps einer Zelle
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US10408735B2 (en) 2019-09-10
EP3042177B1 (fr) 2019-06-05
DE102014200911A1 (de) 2015-04-09

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